Isolation and Characterization of Streptococcus Mutans As Causative Agent of Dental Caries in Gaza Strip and Their Antibacterial Susceptibility Pattern

Al-Azhar University-Gaza Deanship of Postgraduate Studies Faculty of Science Biology Department Biological Sciences Master Program

Isolation and Characterization of Streptococcus mutans as Causative Agent of Dental Caries in Gaza Strip and their Antibacterial Susceptibility Pattern

By: Ibrahim Khalil Ibrahim Abu Ismail B.Sc. Laboratory Medicine

Supervisor Co-Supervisor Dr. Abdallah Bashir Dr. Emad Abou Elkhair Assoc. Prof. of Microbiology Assoc. Prof. of Microbiology

Thesis Submitted in Partial Fulfillment of the requirements for the Master Degree of Science in Biological Sciences

2017

Declaration

"I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree of the university or other institute, except where due acknowledgment has been made in the text".

Ibrahim Khalil Abu Ismail

Acknowledgment

First, I would like to thank my supervisor Dr. Abdallah Bashir for his professionalism, encouragement and enthusiastic. The door to his office was always open whenever I ran into a trouble spot or had a question about my research or writing. He consistently allowed this thesis to be my own work, but steered me in the right the direction whenever he thought I needed it.

Also special thanks to my co-supervisor Dr. Emad Abou Elkhair for his knowledge and interest in this project, overcome all the difficulties aroused during this research, support, encouragement, guidance, endless patience, and for his valuable time which was spent in reviewing and correcting this work.

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Dedication

To my lovely mother,

To my beloved Father,

To my brothers and sisters,

To my wife,

To my lovely children:

(Mona, Noor, Malika, Khalil and Marah),

To my friends and colleagues,

To my teachers and professors,

I dedicate this work

Content Page Declaration II Acknowledgment III Dedication IV Table of contents V List of Figures VIII List of Tables IX List of abbreviations XI Abstract (English) XII Abstract (Arabic) XIV Chapter 1: Introduction and aim of work 1.1 Introduction 2 1.2 Significance 4 1.3 General objectives 4 1.4 Specific objectives 4 Chapter 2: Literature Review 2.1 Dental caries 6 2.1.1 Dental Caries definition according to the Centers for Disease 6 Control and Prevention (CDC) 2.1.2 Dental Caries definition according to the World Health 6 Organization (WHO) 2.1.3 Dental caries definition according Shafer, Hine and Levy 6 2.2 Nature of the Disease (Dental Caries) 6 2.3 Caries mechanism 7 2.4 The factors and sub-factors that influence caries development 8 2.5 Pathogenesis of dental caries 10 2.6 Most common oral bacteria 11 2.6.1 The Streptococcus species 11 2.6.1.1 Streptococcus mutans 12 2.6.1.2 Streptococcus sanguis 14 2.6.1.3 Streptococcus mitis (mitior) 14 2.6.1.4 Streptococcus salivarius 14 2.7 Biofilm formation 14 2.8 Essential oils against dental bacteria 16 2.9 Antibacterial mouth rinses 16 2.10 Antibacterial tooth paste 17 Chapter 3:Materials and Methods 3.1 Study design 19 3.2 Study population 19 3.3 Sample size 19 3.4 Setting of the Study 19 V

3.5 Distribution of samples 19 3.6 Period of the study 19 3.7 Eligibility criteria 20 3.7.1 Inclusion criteria for selection 20 3.7.2 The Exclusion criteria for selection 20 3.8 Ethical and administrative considerations 20 3.9 Chemicals and Reagents 20 3.10 Data collection 23 3.10.1 Sample collection 23 3.10.2 Questionnaire collection 23 3.10.3 Demographic data 23 3.10.4 Clinical data 23 3.11 Preparation of bacterial culture media and Culturing of bacterial 24 isolates 3.11.1 Preparation of culture media 24 3.11.2 Culturing of bacterial isolates 25 3.12 Agar well diffusion method 26 3.13 Enumeration of S. mutans in saliva 27 3.14 Determination of MIC and MBC for essential oils, toothpaste 27 and mouthwashes 3.15 Tube Dilution Method 28 3.16 Statistical analysis 28 Chapter 4: Results 4.1 Demographic characteristics of the study population 30 4.2 Clinical data 31 4.3 Distribution of study population according to the age 34 4.4 Distribution of study population according to the gender 35 4.5 Distribution of study population according to the marital status 35 4.6 Distribution of study population according to the phases of 36 education 4.7 Distribution of study population according to the accommodation 37 4.8 Distribution of study population according to the smoking status 38 4.9 Distribution of study population according to the number of lost 39 teeth 4.10 Distribution of study population according to the types of 40 toothpaste 4.11 Distribution of study population according to the number of 43 times using tooth brushing 4.12 Distribution of study population according to number of visits 44 to the dental clinic yearly 4.13 Distribution of study population according to the brushing teeth 45 before going to bed 4.14 Distribution of study population according to the types of 46 VI

mouthwash used 4.15 Culturing and Enumeration of S. mutans in 1ml of saliva in 47 adult participants 4.16 Results of biochemical tests for suspected S. mutans isolates 49 4.17 Antimicrobial susceptibility 50 4.18 Antimicrobial activity of essential oils against three isolates of 52 S. mutans 4.19 Efficacy of toothpaste against three isolates of S .mutans 55 4.20 Antimicrobial activity of mouth wash against S. mutans 56

4.21 MIC and MBC determination 57 4.21.1 MIC and MBC of nine essential oils (μl/ml) 57 Chapter 5: Discussion 5.1 Clinical data and presence of S. mutans 60 5.2 Enumeration of S. mutans and other oral streptococci 62 5.3 Antibacterial agents and S. mutans 62 5.4 Essential oils and its effect on S. mutans 63 5.5 Tooth paste and mouth rinses and their effects on S. mutans 64 Chapter 6: Summary, Conclusion and Recommendation 6.1 Summary 67 6.2 Conclusion 68 6.3 Recommendation 68 Chapter 7: References 70 Annexes 81

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List of Figures Page

Figure (3.1): Mitis salivarius agar is used to differentiate among 26 species of Streptococcus Figure (4.1): Percentages of S. mutans , S. mitis and S. salivarius in 49 1ml of saliva taken from adult patients. Figure (4.2): Antibiotics susceptibility for S. mutans 51 Figure(4.3): A photograph showing disk diffusion for ampicillin, tetracycline, ciprofloxacin, clindamycin and amoxicillin against S. 51 mutans Figure(4.4): A photograph showing disk diffusion for vancomycin, 52 erythromycin, amoxyclav, oxacillin and doxycyclin against S. mutans Figure(4.5): Clove bud against S. mutans 53 Figure (4.6): Antimicrobial activity of essential oils against S. mutans 55 Figure(4.7):Inhibitory effect of some tooth paste against S. mutans 56 Figure (4.8): Mouthwashes and their effects on S. mutans 57 Figure (4.9): MIC and MBC of essential oils against S. mutans 58

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List of tables page Table (3.1): Commercial Kits, Chemicals, media and Reagents 21 Table (3.2): List of essential oils used in the antimicrobial assay 22 Table (3.3): List of instruments 22 Table(3.4): Type and composition of used media 24 Table (4.1): Demographic characteristics of study population 31 Table (4.2): Clinical data of study population 33 Table (4.3): Distribution of study population according to the age 34 Table( 4.4): Distribution of study population according to the gender 35 Table (4.5): Distribution of study population according to the marital 36 status Table (4.6): Distribution of study population according to the phases of 37 education Table (4.7): Distribution of study population according to the 38 accommodation Table (4.8): Distribution of study population according to the smoking 39 status Table (4.9): Distribution of study population according to the number of 40 lost teeth Table (4.10): Distribution of study population according to the types of 42 toothpaste Table (4.11): Distribution of study population according to the number 44 of times using tooth brushing Table (4.12): Distribution of study population according to the times do 45 you visit the dental clinic yearly Table (4.13): Distribution of study population according to the brushing 46 teeth before going to bed Table (4.14): Enumeration of bacteria in 1ml of saliva on brain heart 47 infusion agar Table (4.15): Enumeration of S. mutans and related species in 1ml of 48 saliva in patients Table(4.16): Results of biochemical tests for suspected S. mutans 50 isolates Table( 4.17): Antibiotics susceptibility for S. mutans 50 Table (4.18): Antimicrobial effect of different plant oils on S. mutans. R 54 equal resistant Table (4.19): Antimicrobial activity of some toothpastes against S. 55 mutans IX

Table (4.20): Inhibitory effect of some mouth washes on the growth of 3 56 selected S. mutans isolates. Table (4.21): MIC of all the nine extracts against S. mutans 58

List of abbreviations

AEP Acquired enamel pellicle BHI Brain heart infusion C. citratus Cymbopogon citratus CDC Centers for Disease Control and Prevention CFU Colony-forming unit EOs Essential oils FDA Food and Drug Administration hrs Hours MBC The minimum bactericidal concentration MIC Minimal Inhibitory Concentration MSA Mitis Salivarius Agar CLSI Clinical and Laboratory Standards Institute NIH National Institutes of Health SPSS Statistical Package for Social Sciences TNTC Too-numerous-to-count VP Voges proskaur Test CPC Cetylpyridinium chloride WHO World Health Organization

Abstract

Isolation and Characterization of Streptococcus mutans as Causative Agent of Dental Caries in Gaza Strip and their Antibacterial Susceptibility Pattern.

Background: Dental caries is a well-known major oral health problem in most countries. The multifactorial etiology of the disease includes multiple bacterial species, S. mutans is the main pathogen associated with the disease. S. mutans is a facultative gram-positive anaerobe commonly found in oral cavity. Objective: The aim of this study is to isolate and characterize S. mutans as potential causative bacteria of dental carries and studying its sensitivity to antibacterial agents and antibacterial mouth rinses from patients visit the dental clinic in Gaza strip. Material and method: Open label experimental study was performed on 300 patients and 300 control visit the dental clinics in Gaza strip. The study was carried out during the period from July 2015 to July 2016. All suspected S. mutans isolates were identified biochemically, tested against ten common used antibiotics by disc diffusion method, studied in vitro for efficacy of nine essential oils, five toothpaste and four mouth rinses.

Results: Among 300 patients tested in this study, 95 showed a positive result for the presence of S. mutans in their saliva and dental caries. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in the age group 20-35 year and in nonsmoker patients. Colgate showed the best antimicrobial activity among 5 different toothpastes. Antibiotic sensitivity test indicated that S. mutans was most susceptible against vancomycin (100%), tetracycline (83.2%), doxycycline (84.2%), ciprofloxacin (81.1%) and amoxyclav (75.8%). Gargarol was found to be the most effective mouth rinse

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against S. mutans. Clove bud, lemon grass and Tea tree were the most effective oils against S. mutans respectively. Conclusion: S. mutans isolates were moderately resistant to antibiotics. Use of plant extracts (essential oils) may be recommended as a supportive or alternative option to conventional formulations. Key words: Streptococcus mutans, Dental caries, Gaza strip, Essential oils, Tooth paste, Mouth rinses.

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الولخص ببلعزبي

عشل وتىصيف البكتيزيب العقذيت الطبفزة الوسببت لتسىص االسٌبى في قطبع غشة وهعزفت هذي هقبوهتهب للوضبداث الحيىيت الوختلفت.

ؼَزجش رسىط االسُبٌ يشكهخ صحُخ فٍ انؼذَذ يٍ انجهذاٌ حىل انؼبنى َظشا نهًسججبد انؼذَذح نهزِ انًشكهخ وانزٍ رزًثم فٍ انؼذَذ يٍ انًسججبد انجكزُشَخ وخصىصبً انجكزُشَب انؼقذَخ انطبفشح وهٍ ثكزُشَب يىخجخ اندشاو الهىائُخ اخزُبسَب, رزىاخذ ػبدح ً فٍ ردىَف انفى ورؼزجش انًسجت انشئُسٍ نزسىط االسُبٌ .

هذفذ هزِ انذساسخ انً ػضل ورىصُف انجكزُشَب انؼقذَخ انطبفشح انؼًضونخ يٍ يشضً رسىط االسُبٌ فٍ قطبع غضح وانزٍ رؼزجش كًسجت سئُسٍ نزسىط االسُبٌ ودساسخ اًَبط يقبويزهب نهًضبداد انحُىَخ انًخزهفخ ويذي فؼبنُخ غسىل انفى انًسزخذو كؼبيم يضبد نهجكزُشَب.

هزِ انذساسخ هٍ دساسخ يفزىحخ اخشَذ ػهً 033 شخص ؼَبَىٌ يٍ رسىط االسُبٌ و033 شخص سهُى ظبهشَبً يًٍ قبيىا ثضَبسح ػُبداد طت انفى و االسُبٌ فٍ قطبع غضح. رى اخشاء هزِ انذساسخ خالل انفزشح انىاقؼخ ثٍُ َىنُى 5302 انً َىنُى 5302, رى رؼشَف خًُغ انؼُُبد انجكزُشَخ انؼًضونخ يٍ خالل انخصبئص انكًُُبء انحُىَخ ورى فحص يذي يقبويزهب نؼششح يٍ انًضبداد انحُىَخ شبئؼخ االسزؼًبل , اَضب رى دساسخ يذي رأثُش ػششح صَىد َجبرُخ و خًسخ يؼبخٍُ اسُبٌ و اسثؼخ غسىل فى ويذي فؼبنُزهى نهحذ يٍ ًَى انجكزُشَب انؼقذَخ انطبفشح خبسج اندسى .

اظهشد َزبئح انذساسخ اٌ 52 يٍ انًشضً ال033 كبَذ انجكزُشَب انؼقذَخ انطبفشح يىخىدح نذَهى فٍ كم يٍ ػُُبد انهؼبة و انؼُُبد انًأخىرح يٍ رسىط االسُبٌ , وقذ كبَذ اغهت هزِ انحبالد يىخىدح نذي انغُش يذخٍُُ يٍ انفئخ انؼًشَخ ي53ٍ-02 سُخ .اظهشد َزبئح انذساسخ اَضبً اٌ يؼدىٌ االسُبٌ كىندُذ كبٌ االكثش فؼبنُخ يٍ ثٍُ انؼًبخٍُ انخًسخ انزٍ اسزخذيذ فٍ انذساسخ ,كزنك اظهشد َزبئح انذساسخ اٌ انجكزُشَب انؼقذَخ انطبفشح كبَذ حسبسخ نهًضبد انحُىٌ فبَكىيُسٍُ ثُسجخ 033% و دوكسُسُكهٍُ ثُسجخ 5..2% و انززشاسُكهٍُ ثُسجخ 20.5% و انسجشوفهىكسبسٍُ ثُسجخ20.0% و ايىكسُكالف ثُسجخ 82.2%. ثبنُسجخ نزبثُشغسىل انفى كبٌ )Gargarol( االكثش فؼبنُخ ضذ انجكزُشَب انؼقذَخ انطبفشح, ايب ثبنُسجخ نزأثُش انضَىد انًسزخذيخ فٍ هزِ انذساسخ ػهً ًَى انجكزُشَب انؼقذَخ انطبفشح اظهش كم يٍ صَذ ثشاػى انقشَفم و صَذ حشُشخ انهًُىٌ وصَذ شدشح انشبٌ فؼبنُخ يًزبصح ػهً انزىانٍ ضذ ًَى انجكزُشَب انؼقذَخ انطبفشح .

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َسزُزح يٍ هزِ انذساسخ اٌ انجكزُشَب انؼقذَخ انطبفشح كبَذ يقبويخ نهًضبداد انحُىَخ ثذسخخ كجُشح واٌ انضَىد انُجبرُخ ًَكٍ اٌ رسزخذو كجذَم ػٍ انًضبداد انحُىَخ او كخُبساد ػالخُخ داػًخ نهًسزحضشاد انزقهُذَخ .

الكلوبث الوفتبحيت: انجكزُشَب انؼقذَخ انطبفشح, رسىط األسُبٌ, قطبع غضح، انضَىد انُجبرُخ, يؼبخٍُ االسُبٌ, غشغشاد انفى .

Chapter 1 Introduction and aim of the work

1. Introduction and aim of the work

1.1 Introduction

Dental caries is a worldwide prevalent costly oral chronic disease, which is also known as dental decay or cavities and characterized as a destruction of the outer layer of teeth (Bagramian et al., 2009). It affects humans throughout their life and is associated not only with pain in the oral cavity but can also cause endocarditis (Banas 2004 ; Strużycka 2014).

Some studies demonstrate that 90% of adults had dental caries in their permanent teeth, and 23% had untreated tooth decay (Beltrán-Aguilar et al., 2005).

Therefore, dental caries represent a global public health problem to be managed by authorities and dental professionals (Bönecker et al., 2012). In the last decades, effective caries-preventive methods have been developed and amended. These methods demonstrate that the chemical control of plaque is an effective strategy to prevent dental caries development (Tsourounakis et al., 2013).

Many factors can be associated with promoting the development of dental caries such as cariogenic bacteria, dietary sugar (Touger-decker and Van Loveren 2003), and exposure to cariogenic effects (Sayegh et al., 2002) as well as diseases affecting the teeth leaving an individual at high risk to dental caries (Ferraz et al., 2012).

In the same context, good oral hygiene play important role in combating the emergence of dental caries and vice versa. In order to take care of his/her teeth, a person needs to have a positive attitude towards dental health. It has been observed that dental neglect is associated with illiteracy amongst low socio economic class and the prevalence of oral diseases are highest amongst them

(Sarkar et al., 2015). About 578 operational taxonomical units (OUT) of bacteria were identified in infants‘ saliva; adults in contrast have 1012 OUT in their saliva (Cephas et al., 2011).

In general, the oral ecosystem is maintained at homeostasis between microbe and ecological niches of mouth such as teeth and tongue (Slavkin 1999). Consumption carbohydrates like the simple sugar glucose leads to changes in oral cavity and allow the overgrowth of acidophilic bacteria known to be damaging to the teeth, resulting in dental caries (Burt and Pai 2001).

The most virulent of oral bacteria are Streptococcus mutans, which have been found to be the initiator of most dental caries (Van et al., 2000; Tanzer et al., 2001; Li et al., 2014, and Ahrari et al., 2015). S. mutans are gram-positive facultative anaerobic cocci shaped bacteria, commonly found in the human oral cavity (Loesche 1996). The pathogenicity of S. mutans depends on their ability to form biofilms on solid surfaces such as teeth (Ahn et al., 2008 and Krzyściak et al., 2014). The pathogenic potential of S. mutans depend also on their ability to metabolize a wide range of sugars, to form biofilm and to create an acidic environment (Argimón and Caufield 2011), and the ability to survive this acidic milieu by production of branched amino acids like valines, leucines and isoleucines (Santiago et al., 2012).

Not only streptococci bacteria colonize the oral cavity, the mouth supports the growth of a wide variety of other microorganisms. These include diverse bacterial species, yeasts, viruses and, on occasions, protozoa (Marsh 1999).

In this work, We aimed to isolate and identify S. mutans from patients suffering of dental caries using conventional methods. We also study the sensitivity of isolated S. mutans to different antibiotics, medicinal plants essential oils and mouth rinses.

1.2 Significance

The main of this study is to isolate and characterize bacterial causative agent which play an important role in dental caries development from patients how visit the dental clinics in Gaza strip and to examine the effectiveness of some antibiotics and the effect of some medicinal plants essential oils, toothpastes and mouth wash present in the local market and recommended by dentists against this bacterial isolate. Also to make a scientific correlation between dental caries and eco-social factors, we designed this work to involve different age groups, genders, marital status, accommodation and education.

1.3 General objectives The aim of this study is to isolate and characterize S. mutans as potential causative bacteria of dental carries and studying its sensitivity to antibacterial agents, medicinal plants essential oils and antibacterial mouth rinses from participants visiting the dental clinic in Gaza strip.

1.4 Specific objectives 1. Isolation and characterization of S. mutans from dental caries and oral cavity of patients visiting the dental clinics in Gaza strip. 2. Studying the antibacterial resistance pattern of S. mutans. 3. Suggestion of oral and teeth protection therapy based on the results achieved from studying sensitivity to antibacterial agents, medicinal plants essential oils and antibacterial mouth rinses.

Chapter 2 Literature Review

2. Literature review

2.1 Dental caries

2.1.1 Dental Caries definition according to the Centers for Disease Control and Prevention (CDC)

Dental caries or cavities, more commonly known as tooth decay, are caused by a breakdown of the tooth enamel. This breakdown is the result of bacterial growth on teeth that breakdown foods and produce acid that destroys tooth enamel and results in tooth decay (Dye et al., 2007).

2.1.2 Dental Caries definition according to the World Health Organization (WHO)

Dental Caries is defined as a localized, post-eruptive, pathological process of external origin involving softening of the hard tooth tissue and proceeding to the formation of cavity (WHO 1972).

2.1.3 Dental caries definition according Shafer, Hine and Levy

Dental caries is a complex microbial disease of the calcified tissues of the teeth, characterized by demineralization of the inorganic portion and destruction of the organic substance of the tooth. It is the most prevalent chronic disease affecting the human race (Shafer et al., 2009).

2.2 Nature of the Disease (Dental Caries) A caries lesion is the clinical appearance of a pathological process that may have been occurring on the tooth surface for months or years. Caries lesions are often confused for the disease process. Reducing the prevalence of caries lesions through, for example, topical application of fluoride may have little or no effect on the disease process. Caries lesions result from the interaction of the bacteria,

which colonize or infect the tooth surface with constituents of the diet, usually sucrose. The appearance of dental plaque is the first overt clinical evidence of this interaction. Nevertheless, the processes involved in the initiation and formation of plaque are complex and diverse. Plaque is initiated by the formation of a salivary pellicle on the tooth surface. Although most attention has been focused on the mammalian constituents of pellicle, there is unequivocal evidence that bacterial constituents are present from the earliest stages of formation (Kim et al., 2013).

2.3 Caries mechanism

Dental caries is a simple process in concept, but complicated in detail. In outline, the caries mechanism can be described as follows: (1) Acidogenic (acid-producing) oral plaque bacteria ferment carbohydrates that are taken into the mouth, thereby producing organic acids, including lactic, formic, acetic, and propionic acid. (2) These acids diffuse into the enamel, dentin, or cementum, partially dissolving the mineral crystals. (3) Mineral (calcium and phosphate) diffuses out of the tooth, leading eventual to cavitation if the process continues. (4) Demineralization can be reversed by calcium and phosphate, together with fluoride, diffusing into the tooth and depositing a new veneer on the crystal remnants in the non cavitated lesion (this is remineralization). (5) The new mineral crystal surface is much more resistant to acid as compared with the original carbonated hydroxyapatite mineral. (6) The process of demineralization and remineralization generally occurs numerous times daily, leading either to cavitation, to repair and reversal, or to maintenance of the status quo. In root caries, the same mechanism occurs as outlined above, initially causing demineralization and exposure of the collagen fibril (Featherstone 2004).

2.4 The factors and sub-factors that influence caries development: Dental Caries A Multi-factorial Disease The development of caries is dependent on the interaction of four primary factors. These are a host (tooth surface), a substrate (food), the presence of oral bacteria, and time. Caries will not develop if any of these four primary factors are not present (Marcotte and Lavoie1998). Each of the four primary factors can be further divided into sub-factors that also influence the likelihood of caries: 1. Host (tooth surface): The sub-factors that influence caries development are age (the enamel of the deciduous teeth of children is more susceptible to acid demineralization), if fluoride has been used, tooth morphology (which varies within the mouth and from person to person), root surface exposure due to gum recession, nutrition (if tooth strengthening nutrients are consumed), and saliva flow rate and buffering capacity. A tooth is more susceptible to caries if it has less acid resistant enamel due to age or low fluoride intake, or if the roots have been exposed by gum recession. Caries risk is also higher if the diet is low in nutrients (such as magnesium and vitamin D) that are necessary for healthy tooth development, and/or when an individual‘s saliva flow rate is low or has a low buffering capacity. Pit-and-fissure demineralization is more likely to develop in teeth with numerous and exaggerated grooves. Teeth are less prone to caries activity in situations where tooth enamel has been strengthened by fluoride, a diet of tooth strengthening nutrients is consumed, and/or the buffering capacity of saliva is high ( Moynihan and Petersen 2004).

2.Substrate (food): The sub-factors that influence caries development are oral clearance (if food is retained or not in the mouth after eating), oral hygiene (if, after eating, food is actively removed with a sharp instrument such as a toothpick), eating frequency, food detergency (if consumed food can clean

teeth), consumption of carbohydrates, and the cariogenicity of consumed carbohydrates (sucrose is more cariogenic than glucose and fructose). When food is retained in the mouth and not actively removed after eating, is consumed more frequently, and/or more sugars, sucrose containing foods, and sticky foods (like toffee) are consumed, there is higher risk of caries. On the other hand, when remaining food particles are actively removed after eating, food is consumed less frequently, fewer sugars, sucrose-containing foods, and sticky foods are consumed, and/or more tooth cleaning foods (like apples) are eaten, the likelihood of caries is lower ( Selwitz et al., 2007).

3.Oral bacteria: The development of caries depends on microbial load (how much bacteria is present), plaque composition (with some types of plaque microbes being more cariogenic than others), plaque acidogenicity (how much acid can be produced by the plaque that is present), plaque acidoduricity (how well plaque can survive in acidic conditions), oral hygiene (how often microbial load is reduced by brushing or prophylaxis), and if fluoride is present in plaque. The likelihood of caries development is higher when microbial load is high as indicated by excessive plaque, when more caries-linked bacteria are present in plaque, when plaque produces more acid, when more plaque bacteria can survive in acidic conditions, and/ or when plaque is not regularly removed by brushing. The odds that caries will develop are lower when the microbial load is low as indicated by little plaque, present plaque has fewer bacteria associated with caries or that can withstand very acid conditions, plaque acid production is low, and/or plaque is regularly removed by brushing or flossing (Kolenbrander et al., 2002).

4. Time: While the shift in microflora can occur over a fairly short period, a significant amount of time is needed for demineralization to lead to the development of whitespot and/or carious lesions. Acid production does not

instantly trigger tooth decay, and in the early stages, remineralization can restore enamel, keeping the effects of dental caries at bay. In summary, bacterial fermentation of consumed sugars produces acid in the tooth‘s immediate environment. This acid demineralizes tooth enamel, and over time, this dissolution of tooth structure leads to the development of carious lesions. Because the combination of factors and sub-factors include unavoidable situations, dental caries can be very difficult to prevent (Higham 2010).

2.5 Pathogenesis of dental caries About 700 different bacteria species have been identified from the human oral microbiome (Marsh et al., 2009). In the pathogenesis of dental caries an important role play cariogenic bacteria, i.e. oral streptococci, especially of group mutans and lactic acid bacteria (Lactobacillus spp.). It is believed that bacteria of the species S. mutans is the main factor that initiates caries and very important factor of enamel decay. The bacteria of the genus Lactobacillus are important in further caries development, especially in the dentin. S. mutans and lactobacilli are characterized by the ability to grow in an acid environment and the property of rapid metabolism of sugars supplied in the diet to organic acids, including lactic acid (Byun et al., 2004 and Marsh 1999). The microbial community from dentinal lesions is diverse and contains many facultatively- and obligately-anaerobic bacteria belonging to the genera Actinomyces, Bifidobacterium, Eubacterium, Lactobacillus, Parvimonas and Rothia. Streptococci are recovered less frequently (Marsh 2009). Caries can also be caused by other bacteria, including members of the mitis, anginosus and salivarius groups of Streptococci, Propionibacterium, Enterococcus faecalis and Scardovia. (Tanzer et al., 2001 and Wade 2013). In molecular studies using 16S rRNA analysis has been demonstrated, that the predominant microbes in deep caries lesions were S. mutans and genus

Lactobacillus but also included the genera Prevotella, Selenomonas, Dialister, Fusobacterium, Bifidobacterium and Pseudoramibacter (Munson et al., 2004 and Chhour et al., 2005). A large number of scientists they reported that genera associated with dental caries in both primary and permanent dentitions are Streptococcus including S. mutans, S. sanguinis and non-S. mutans streptococci, Veillonella, Actinomyces, Bifidobacterium, Lactobacillus, Propionibacterium, and Atopobium. In other study, has been shown that in plaque significantly associated with dental caries are the genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia and Thiomonas (Ling et al., 2010).

2.6 Most common oral bacteria The oral cavity is inhabited by microflora composed of a very wide spectrum of organisms, bacteria comprising the dominant part (approx. 70% Streptococci), there are also smaller numbers of virae, mycoplasmates, yeasts and protozoa. The composition of the oral microflora changes with age and is influenced by host factors, including the eruption and the loss of teeth, and the insertion of partial or complete dentures, the level of oral hygiene, saliva quality and quantity, perorally administered medicaments and the general state (age, immunity system, illness) (Kagermeier et al., 2000) .

2.6.1 The Streptococcus species Streptococci comprise the majority of oral microorganisms. They are Gram- positive non-sporulating cocci of circular to ovoid shape, facultative anaerobes (some requiring CO2), catalase negative, homo-fermentative chemo- organotrophes with complex nutritional requirements. They are typically assembled into chains and are widespread in nature. Forty of the currently known species of Streptococci are found predominantly on the mucosa surfaces of humans and animals, the upper respiratory tract, on skin, the gastro-intestinal

tract, they can also be found in soil, dairy products, other foods and plants. It has been possible to isolate Streptococci from all locations in the oral cavity. On average, they are represented by 28% in tooth plaque, 29% in the gingival crevice, 45% on the tongue, and 46% in saliva. Insoluble extracellular polymers play an important role in colonizing exposed tooth surfaces by some oral streptococci. Polymer production is also a key test in identifying schemes of these organisms (Cowman et al., 1977).

2.6.1.1 Streptococcus mutans S. mutans (Phylum Firmicutes) is one member of the streptococcal group known as the ―mutans‖ group .The mutans group is one of five groups in the ―viridans group,‖ which also includes the ―anginosus group,‖ ―bovis group,‖ ―mitis group,‖ and ―salivarius group.‖ All viridans streptococci are either alpha- hemolytic or non-hemolytic; Gram-positive cocci typically found in the mouth, upper respiratory tract, and urogenital tract of humans. Members of the mutans group are the most common cause of sub-acute endocarditis in patients with existing heart valve problems or prosthetic heart valves. They are also responsible for bacteremia following dental or urogenital invasive procedures and in immunosuppressed patients undergoing chemotherapy and bone marrow transplantation. Clinically, the most common encounter with oral streptococci is in the dentist‘s chair. Several of these bacterial groups are capable of hydrolyzing sucrose and forming dental plaque, which in turn provides the anaerobic environment ideal for fermentation. Acids produced by this fermentation and that of certain Lactobacilli erodes the tooth enamel and is responsible for the formation of dental caries. The spread of drug resistant pathogens is one of the most serious threats to successful treatment of microbial diseases. Down the ages, essential oils and other extracts of plants have evoked interest as sources of natural products. They

have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases (Tepe et al., 2004). Peppermint, tea tree and thyme oils can act as an effective intracanal antiseptic solution against oral pathogens (Thosar et al., 2013). Cinnamon oil, Lemon oil, Cedar wood oil, Clove oil and eucalyptus oil exhibit antibacterial property against S. mutans (Chaudhari et al., 2012).

S. mutans is the primary etiologic agent of dental caries .and multiple characteristics contribute to its successful colonization, persistence, and virulence in the dynamic environment of the human oral cavity. Pathogenesis by S. mutans requires efficient biofilm formation on the tooth surface, as well as the fermentation of carbohydrates to organic acids that directly cause demineralization of tooth enamel. Acid tolerance, or aciduricity, which is the ability to grow and metabolize nutrient at low pH, is also a defining characteristic of cariogenic microorganisms. Oral microbial communities are one of the most complex microbiomes of the human body. To date, hundreds of different taxa have been identified using DNA-based methodologies, and many species that are abundant in the oral flora display remarkable degrees of genetic and phenotypic heterogeneity, which undoubtedly has a profound effect on the pathogenic potential of the oral microbiome (Kim et al., 2013).

S. mutans is a Gram-positive bacterium, which plays a key role in the formation of the dental plaque biofilm as an early coloniser (it produces adhesions which attach the organism to the acquired pellicle of the teeth) and is the most important bacterium in the formation of dental caries (Loesche 1986). The structure of S. mutans can be seen in the representative Gram-positive bacterium but note that S. mutans do not have flagella, but do have pili (Kline et al., 2010). S. mutans are Gram-positive ovoid cocci, that typically occur in pairs or chains,

are aciduric (grow well in acid medium) and acidogenic (produce acid) and are non-motile facultative anaerobes that grow optimally at 37°C (Wilson 2008).

2.6.1.2 Streptococcus sanguis Studies of this strain have proven this to be one of the primary colonizers of the clean tooth surface. These strains produce extracellular insoluble and soluble glucanes from sucrose. They firmly adhere to the surface of epithelial cells, thus helping to shield the surface from other bacterial species, that can be inhibited by both occupying receptors and bacteriocine production (Marsh et al., 2009).

2.6.1.3 Streptococcus mitis (mitior) This is probably the most frequently isolated species from tooth plaque. Some strains produce extracellular polymers from sucrose. It colonizes hard dental tissue as well as mucous membranes, especially the cheeks and tongue. Although S. mitis may cause caries, in most cases it is considered a harmless member of the oral microflora (Mei and Busscher 1996).

2.6.1.4 Streptococcus salivarius S. salivarius strains can be isolated from all locations in the mouth, though they prefer to colonize epithelial surfaces. S. salivarius produces an extracellular polymer levane from sucrose. This is a highly unstable polymer that can be metabolized by other microorganisms (Marsh et al., 2009) .

2.7 Biofilm formation The microorganisms that comprise the human dental biofilm co-aggregate by a complex and dynamic process to form a multi-species community, biofilm. On hard surfaces (teeth), biofilms are typically several cell layers thick and can measure up to several hundred micrometers (Kolenbrander 2000).

There is much that we do not understand about the factors governing biofilm formation, it is clear that the process is complex and involves contributions made by a very large number of bacterial species. Biofilm formation on tooth surfaces is initiated by what are known as ‗pioneer colonisers comprised mainly of mitis group members such as S. gordonii, S. sanguinis and S. mitis. The establishment of these species allows the early colonisers including S. mutans, Veillonella spp. and bridging function provided by Fusobacteria spp. to co- colonise the biofilm. Maturation of the biofilm involves addition of late colonisers comprised primarily of anaerobic, gram-negative bacteria (Kolenbrander et al., 2006 and Zijnge et al., 2010).

S. mutans are the most cariogenic pathogens as they are highly acidogenic, producing short-chain acids which dissolve hard tissues of teeth. They metabolize sucrose to synthesize insoluble extracellular polysaccharides, which enhance their adherence to the tooth surface and encourage biofilm formation. The reactions are catalyzed by three isozymes of glucosyltransferases (Islam et al., 2007). S. mutans is able to metabolite a number of sugars and glycosides such as glucose, fructose, sucrose, lactose, galactose, mannose, cellobiose, glucosides, trehalose, maltose and a previously unrecognized, group of sugar-alcohols. In the presence of extracellular glucose and sucrose, S. mutans synthesizes intracellular glycogen-like polysaccharides (IPSs) (Peterson et al., 2011). S. mutans produces also mutacins (bacteriocins), what is considered to be an important factor in the colonization and establishment of S. mutans in the dental biofilm(Merritt and Qi 2012).

2.8 Essential oils against dental bacteria Essential oils (EOs) have aroused attention among the naturally occurring bioactive agents with promising antimicrobial activity (Bassolé and Juliani 2012). EOs are a mixture of volatile constituents produced by aromatic plants as secondary metabolites, as a protective mechanism against predators, microorganisms or weather adversities (Bakkali et al., 2008). The antimicrobial activity of EOs against dental bacteria - especially against the tooth-decay causing bacteria Streptococcus pyogenes and Streptococcus mutans. EOs are capable of inhibiting the growth of these bacteria as well as the formation of biofilms. In various cases the potency of chlorohexidine was found to be even lower than the efficacy of the Eos (Rasooli et al.,2008). Therefore, the application of EOs is recommended in products which prevent caries (Maggi et al., 2009). Essential oils distilled from members of the genus Lavandula have been used both cosmetically and therapeutically for centuries with the most commonly used species being L. angustifolia, L. latifolia, L. stoechas and L. x intermedia. Although there is considerable anecdotal information about the biological activity of these oils much of this has not been substantiated by scientific or clinical evidence. Among the claims made for lavender oil are that is it antibacterial, antifungal, carminative (smooth muscle relaxing), sedative, antidepressive and effective for burns and insect bites (Cavanagh and Wilkinson 2002).

2.9 Antibacterial mouth rinses Mouth rinsing was first practiced as an oral hygiene measure in Chinese medicine (Barnett 2006). A well-documented scientific and clinical basis for the use of therapeutic antimicrobial mouth rinses, however, was recorded

relatively recently, in the 1960s. A plaque- and calculus-inhibiting effect of the quaternary compound cetylpyridinium chloride CPC (Schroeder et al.,1962). CPC is a cationic surface-active agent and has a broad antimicrobial spectrum, with rapid killing of gram-positive pathogens and yeast in particular (Pitten and Kramer 2006).

2.10 Antibacterial tooth paste Standard toothpaste formulas generally contain a combination of fluoride and detergents, enhancing the efficacy of biofilm control(Davies 2008 and Marsh 2010).The addition of different antimicrobial agents has been suggested as a potential method for reduction, control and prevention of the accumulation of cariogenic and periodontopathogenic microorganisms (Prasanth 2011; Maltz and Beighton2012). Toothpaste is a gel dentifrice used with a toothbrush as an accessory to clean and maintain the aesthetics and health of teeth. Toothpaste is used to promote oral hygiene (Wright et al.,2014). Triclosan, an antibacterial agent, is a common toothpaste ingredient in the United Kingdom. Triclosan or zinc chloride prevents gingivitis and, according to the American Dental Association, helps reduce tartar and bad breath. Herbal toothpastes contain baking soda, aloe, eucalyptus oil, myrrh, plant extract, and essential oils. Various Oral microfloraincludes most commonly E. coli and C. albicans(Hernandez 2002).

Chapter 3 Materials and Methods

3.1 Study design

Open label experimental study was performed on 300 patients visit the dental clinic in Gaza strip. The bacterial isolates (S. mutans) were conventional identified and tested for antimicrobial susceptibility using disk diffusion method. Screening for the antibacterial activity of different medicinal plants essential oils, toothpaste and mouthwashes were also performed.

3.2 Study population

Patients having dental caries and suffer of mouth infection (diagnosed by a dentist) as well as control persons having healthy teeth and healthy teeth gums.

3.3 Sample size

This open label experimental study will include 300 patients and 300 control.

3.4 Setting of the Study

The analytical part of this study was performed in the laboratory of microbiology in the biology department at Al-Azhar University-Gaza.

3.5 Distribution of samples

Samples were collected at dental clinics of the military medical services in Gaza strip.

3.6 Period of the study

The study was carried out during the period from July 2015 to July 2016.

3.7 Eligibility criteria

3.7.1 Inclusion criteria for selection:

 Age more than 20 years.( for cases and control )

3.7.2 The Exclusion criteria for selection:

 Marked intra oral soft tissue pathology.  Patients with history of taking antibiotics three months prior or during the course of study.  Medically compromised patients.

3.8 Ethical and administrative considerations

Approval letter was obtained from the Helsinki Committee in Gaza strip. Consent form to participate in the study was obtained from the participants. Every participant had been provided with a full explanation about the intended study. Also, assurance of voluntary participation was maintained.

3.9 Chemicals and Reagents

All chemicals and reagents in this study were of analytical grade. Bacteriological media were purchased from Hi Media Company (India) and Sigma Aldrich Company (Germany). Other disposable materials like Petri dishes, cotton swabs and plastic loops were purchased from local distributors in Gaza city.

Commercial Kits, Chemicals, media and Reagents

Table (3.1): Commercial Kits, Chemicals, media and Reagents

Chemicals and kits Absolute ethanol Anaerobic gas bags Brain heart- infusion agar Bile Esculin Agar Gram stain kit Brain heart infusion broth Mitis salivarius agar Mannitol salt agar Antibiotics Abbreviation and Antibiotics Abbreviation and potency potency

Amoxicillin AMX (30 µg) Amoxicillin- AMC (30 µg) clavulanic acid Ampicillin AMP (2 µg) Clindamycin CD (2 µg) Ciprofloxacin CIP (5 µg) Doxycycline DO (30 µg) Erythromycin E (15 µg) Oxacillin OX (1 µg) Tetracycline TE (30 µg) Vancomycin VA (5 µg) Mouth rinses Gargarol Garosept Iodocare Septoral Tooth paste B-white Colgate Paradontax Sensodyne Signal

List of essential oils used in the antimicrobial assay

Table (3.2): List of essential oils used in the antimicrobial assay

Local Arabic Commercial name Scientific name Family Sources name Peppermint oil Mentha piperta Eugenia Labiateae Germany انؼُُبع انفهفهٍ Clove bud oil Caryophyllata part bud Myrtaceae Germany انقشَفم Lemon grass oil Cymbopogon citratus Poaceae Germany حشُشخ انهًُىٌ

Thyme oil Thymus sature iodes Labiateae Germany انضػزش coss Ginger oil Zingiber officinale Zingiberaceae Germany انضَدجُم

Sage Salvia officinalis Lamiaceae Germany يشًَُخ

Tea tree Melaleuca alternifolia Myrtaceae Germany يالنىكب

Cinnamon leaf Cinnamomum Lauraceae Germany قشفخ zeylanicum

Rosemary Rosmarinus officinalis Lamiaceae Germany إكهُم اندجم

List of instruments

Table (3.3): List of instruments

Instrument Manufacturer (country) Autoclave Tomy (Japan) Deep freezer Sanyo (Japan) Incubator Sanyo (Japan) Optical microscope Olympus (Japan) Refrigerator Sanyo (Japan) Safety cabinet Dalton (Japan)

3.10 Data collection

3.10.1 Sample collection

All saliva sample and dental caries taken from patients and controls were collected in sterilized test tube and sent to the laboratory within 2 hours.

3.10.2 Questionnaire collection

Two simple questionnaire were constructed and conducted, one in Arabic language for collecting of important and reasonable demographic data, and the other in English and Arabic languages that constructed to collect relevant dental clinical data about dental caries.

3.10.3 Demographic data

Interview with patients was used for filling a simple questionnaire that designed for matching the study need (Annex 1).

The questionnaire includes questions on socio-demographic data (age, gender, marital status, education, accommodation and smoking).

3.10.4 Clinical data

The dentists filled a short questionnaire about states and clinical features of the patient after examining the patient properly (Annex 1 and 2).

3.11 Preparation of bacterial culture media and Culturing of bacterial isolates

3.11.1 Preparation of culture media

All bacterial culture media used in the present study were prepared according to the manufacturer‘s instructions.

Table 3.4 showed the type and composition of media used in this study.

Table(3.4): Type and composition of used media

Blood Agar media

The media is enriched, differential, used to isolate fastidious organisms such as streptococci and detect hemolytic activity

Preparation of blood agar base

 Prepared blood agar was transferred to a 50 °C water bath.  The agar cooled to 50°C, sterile blood agar aseptically added and mixed well gently, formation of air bubbles was avoided.  The Blood was warmed to room temperature.  15 ml amounts were dispensed to sterile petri plates aseptically  The plates were store at 2-8 °C, preferably in sealed plastic bags to prevent loss of moisture. The shelf life of thus prepared blood agar is up to four weeks Brain heart infusion agar

 52 grams of brain heart infusion agar were dissolved in 1000 ml distilled water  Heated to boiling till the medium dissolved completely, the medium was  Sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Mixed well before pouring Mitis Salivarius Agar Base

Mitis Salivarius Agar is recommended for the isolation of oral streptococci including S. mutans

 90.07 grams of Mitis Salivarius Agar Base were suspended in 1000 ml distilled water

 Boiled to dissolve the medium completely  Dispensed and sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes  Cooled to 50-55°C and 1 ml of sterile 1% Potassium Tellurite Solution (FD052) were added to the medium. Mannitol Salt Agar

 111.02 grams of mannitol salt agar were suspended in 1000 ml distilled water and boiled to dissolve the medium completely  The mixture was sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes  Cooled to 45-50°C. Bile Esculin Agar Base

 31.75 grams were suspended in 500 ml distilled water.  Boiled to dissolve the medium completely  Cooled to 45-50°C and add rehydrated contents of 1 vial of Esculin (FD050). Mixed and dispensed into tubes or flasks as desired Sterilized by autoclaving at 15lbs pressure (121°C) for 15 minutes.

3.11.2 Culturing of bacterial isolates

All samples, whether from saliva or from decay has been cultivated on BHI agar and MSA. All agar plates were cultivated in a sealed container containing a bag of carbon dioxide at 37°C for 48 hrs. Colony forming units were then counted and the colonies on MSA were selected for further identification via staining and biochemical tests. The sensitivity of S. mutans against antibacterial agents and toothpaste were also determined(Woo et al., 2003).

S.mutans

S.salivarius S.mitis

Figure (3.1): Mitis salivarius agar is used to differentiate among species of Streptococcus

Bacterial isolates identification

Suspected S. mutans colonies on Mitis Salivarius Agar Base (Fig 3.1) were submitted to cultural (Mannitol salt agar and Bile Esculin agar) and biochemical testes identification, including Voges Proskaur, Catalase test, Sorbitol, Mannitol and Sucrose fermentation(Essam et al., 2014).

3.12 Agar well diffusion method

Agar well-diffusion method was followed to determine the antimicrobial activity. BHI agar plates were inoculated with saliva sample and dental caries washing using sterile cotton swabs , five wells (5 mm diameter and about 2 cm a part) were made in each of these plates using sterile cork-borer. Stock solution of each essential oils, toothpastes and mouthwashes were prepared(Balouiri et al.,2016).

About 50 μl of different stock solution were added using sterile micropipette into the wells and allowed to diffuse for 2hrs at refrigerator. The inoculated

plates and carbon dioxide bags were placed in anaerobic jar and were incubated at 37°C for 24 hrs. The diameter of the inhibition zones (mm) were measured and the activity index was calculated (Holder and Boyce 1994).

3.13 Enumeration of S. mutans in saliva

Materials:

Four sterile test tubes that contain 0.9ml of sterile normal saline

0.1ml saliva sample from patients and controls

Procedure:

100µl of saliva sample was mixed in 0.9ml sterile saline. Serial dilutions were -1 -2 -3 -4 prepared as followed: 10 , 10 , 10 and 10 . 0.1 ml of each dilution was spread on 2 different agar media: BHI agar and MSA and incubated at 37 °C in anaerobic jar containing carbon dioxide bag for 48 hours. Developed colony forming units were counted on each agar media (Gallez et al., 2000 and Ogawa et al., 2012).

3.14 Determination of MIC and MBC for essential oils, toothpaste and mouthwashes:

The MIC (Minimal Inhibitory Concentration) of a bacterium to a certain antimicrobial agent gives a quantitative estimate of the bacterial susceptibility to this antimicrobial agent.

MIC is defined as the lowest concentration of antimicrobial agent required to inhibit growth of the organism. The principle is simple: Agar plates, tubes or microtitre trays with two-fold dilutions of antibiotics are inoculated with a standardized inoculum of the bacteria and incubated under standardized

conditions following CLSI guidelines. The next day, the MIC is recorded as the lowest concentration of antimicrobial agent with no visible growth.

MIC-determination performed as agar dilution is regarded as the gold standard for susceptibility testing (CLSI 2011).

3.15 Tube Dilution Method :

It‘s used to obtain Minimum Inhibitory Concentration (MIC). The microbial inoculum was standardized at 0.5McFarland, 100μl of bacteria were aseptically introduced of S. mutans were added to each tubes that contains 5ml of sterile Brain heart infusion broth with different concentration of 9 essential oils, 5 tooth pastes and 4 mouth washes . After the tube of suspensions were done, all tubes were incubated at 37ᵒC for 24-48 hours. Assess the result of incubation by looking at the tube blurriness then MIC can be determined. Three randomly selected isolates of S. mutans were chosen for the purpose of determining the MIC (Nwaokorie et al., 2010).

To obtain Minimum Bactericidal Concentration (MBC), the suspension of each tubes is streaked on the Blood agar plate as many as 0,1 ml. Then it has incubated in anaerobe environment at 37°C for 24-48 hours. Colony forming unit can be seen after the incubation (CLSI 2011).

3.16 Statistical analysis

Data generated from this work was tabulated, entered into Microsoft excel sheets and uploaded to SPSS (Statistical Package for Social Sciences version (22) software. Frequencies cross tabulation and appropriate statistical tests as Chi-square test, fisher exact test and others were performed.

Chapter 4

RESULTS

4.1 Demographic characteristics of the study population

This Part represents the findings of statistical data analysis.

Six hundred participants were included in this study; they were divided into 300 apparently healthy and 300 participants (suffering from dental caries). The participants aged between 20 and 65 year and were divided into three age groups; the first age group was 20-35 years old (40.7%), the second was 36-50 years old (32.8 %) and the third age group was 51-65years old (26.5%).

The distribution of study participants showed that 331 (55.2%) were male and 269 (44.8%) were female; 137 (22.8%) of them were primary educated ,193 (32.2%) were secondary educated and 270 (45.0%) were highly educated; 427 (71.2%) of the study population were married wile 173 (28.8%) were not married (single, widowed and divorced), 226 (37.7%) were smokers and 374 (62.3%) were non- smokers, according to the governorate distribution of samples were equal for each governorate (120 participant from each one).

Table (4.1): Demographic characteristics of study population

Variable No. Of prticipants (600) Percentage (%) Age 20-35 244 40.7 36-50 197 32.8 51-65 159 26.5

Gender Male 331 55.2 Female 269 44.8 Education level Primary 137 22.8 Secondary 193 32.2 Higher education 270 45.0 Matrial status Single 141 23.5 Married 427 71.2 Widowed 23 3.8 Divorces 9 1.5 Smoking status Smokers 226 37.7 Non smokers 374 62.3 Gevornorates Rafah 120 20.0 Khan Younes 120 20.0 Middle 120 20.0 Gaza 120 20.0 North of Gaza 120 20.0

4.2 Clinical data

Table (4.2) represent the distribution of study population according to the loosing of teeth, cleaning of teeth, presence and absence of S. mutans as suspected causative agent of dental caries in saliva as well as in cares, toothpaste and mouthwash and finally visiting of dental clinics

Table (4.2) shows that 239 (39.8%) have no loosing teeth, 187 (31.2%) have at least one loosing tooth, 111 (18.5%) of them were having two loosing teeth and 63 (10.5%) were having more than two loosing teeth.

142 (23.7%) of the study population didn't using tooth brush for teeth cleaning , while 167(27.8%) were using tooth brush once daily, 192 (32.0%) were using tooth brush twice daily and 87 (14.5%) were using tooth brush for three times daily.

According to the presence or absence of S. mutants, Table (4.2) shows that Ninety five(15.8%) were having S. mutants in saliva and dental carries, 205 (34.2%) were negative for presence of S. mutants in dental carries and saliva, while we did not find S. mutans in the saliva sample of all healthy persons (control) participated in this study (300 persons, 50.0%)

Most of study population did not used any mouthwash except for 28 (4.7%).

Three hundred eighty nine (64.8%) of study population were brushing teeth before going to bed versus 211(35.2%) did not.

Distribution of study population according to the visiting dental clinics shows that 150 (25.0%) didn't visit dental clinics, 197 (32.8%) were visited dental clinics once yearly, 166 (27.7%) were visited dental clinics twice yearly and 87(14.5%) were visited dental clinics more than two times yearly.

According to the type of toothpaste used 142 (23.7%) did not use any toothpaste, 98 (16.3%) were using Sensodyne, 123 (20.5%) were using Colgate, 69 (11.5) were using Paradontax, 135 (22.5%) were using Signal and 33 (5.5%) were using B.white.

Table (4.2): Clinical data of study population

Variable No. Of participants (600) Percentage (%)

Presence (+ve) or absence (-ve) of S. mutans in pat (300) and control (300) +ve (patients) 95 15.8

-ve (patients) 205 34.2

-ve (control) 300 50 Visiting of dental clinic in year (before this questionnaire) Non 150 25.0 once yearly 197 32.8 twice yearly 166 27.7 more than 2 times yearly 87 14.5 No. lost teeth Non 239 39.8 One 187 31.2 Two 111 18.5 More than Two 63 10.5 No of time using tooth brushing Non 142 23.7 Once daily 167 27.8 Twice daily 192 32.0 three times a day 87 14.5 more than 3 times a day 12 2.0 Brushing teeth before sleeping Yes 389 64.8 No 211 35.2 Type of used teeth paste Sensodyne 98 16.3 Colgate 123 20.5 Paradontax 69 11.5 Signal 135 22.5 B.white 33 5.5 NON 142 23.7 Type of used mouth wash Gargarol 1 0.2 Septoral 27 4.5 Garosept 0 0.0 Iodocare 0 0.0 Non 572 95.3

4.3 Distribution of study population according to the age

Among 300 patients tested in this study, 95 showed a positive result for the presence of S. mutans in their saliva and dental caries, while 205 were negative for the presence of S. mutans. Table (4.3) shows that 49 patients (33.1%) of the first age (20-35 year) group were positive for the presence of S. mutans in their caries and saliva, while 99 patients in the same age group (66.9%) were negative for S. mutans in dental caries and saliva.

Twenty five patients (29.1%) of the second age (36-50 years) group were positive for S. mutans in their dental caries and saliva, while 61 persons (70.9%) of the second age group were negative diagnosed for S. mutans in dental caries and saliva. Twenty one patients (31.8%) in the last age (51-65 year) group were positive diagnosed for S. mutans in dental caries and saliva, while 45 patients (68.2%) in the same age group were negative for S. mutans in dental caries and saliva.

These results shows statistically significance between age and presence of S. mutans in dental caries and saliva (P = 0.027).

Table (4.3): Distribution of study population according to the age

Positive or negative for the presence of S. Age group mutans Patients Chi-Square P Value 20-35 36-50 51-65 +ve(n=95) .5 25 21 % (33.1%) (29.1%) (31.8%) -ve(n=205) 99 61 45 % (66.9%) (70.9%) (68.2%) Total(n=300) 148 (100%) 68(100%) 66(100%) 5.098 0.027 Control 20-35 36-50 51-65 Total(n=300) 96 111 93 (100%) (32.0%) (37.0%) (31.0%)

4.4 Distribution of study population according to the gender Table (4.4) shows the presence of S. mutans in male and female patients. 62 of male patients (31.6%) had S. mutans in their dental caries and saliva, while 134 of male patients (68.4%) were negative for the presence of S. mutans on saliva and dental caries. All 135 healthy male (45.0%) were negative of S. mutans in saliva. 33 female patients (31.7%) were positive for S. mutans in their dental caries and saliva, while 71 female patients (68.3%) had no S. mutans in their saliva and dental caries. All 165 healthy females (55.00%) were negative of S. mutans in saliva.

Table 4.4 shows that there were statistically significance between the gender and the present of S. mutans (P= 0.007).

Table( 4.4): Distribution of study population according to the gender

Positive or negative for the presence of S. mutans Gender Patients Chi-Square P Value Male female +ve(n=95) 62 33 % (31.6%) (31.7%) -ve(n=205) 134 71 % (68.4%) (68.3%) Total(n=300) 196(100%) 104(100%) 5.054 0.007 Control Male Female Total(n=300) 135 165 (100%) (45.0%) (55.0%)

4.5 Distribution of study population according to the marital status

Table (4.5) shows the presence of S. mutans according to the marital status. Ten (24.4%) of single patients had S. mutans in their dental caries and saliva, while 31(75.6%) of single patients were negative for S. mutans on saliva and dental

caries. 82 patients (33.1%) of married patients had S. mutans in their dental caries and saliva, while 166 (66.9%) of married patients were negative for S. mutans on saliva and dental caries.

Two participants (25.0%) of widowed patients had S. mutans in their dental caries and saliva, while 6 (75.0%) of widowed patients were negative for S. mutans on saliva and dental caries.

One patient (33.4%) of divorced patient had S. mutans in their dental caries and saliva, while 2 (66.6%) of divorced patients were negative for S. mutans on saliva and dental caries.

Table (4.5) shows that there were statistically significance between the marital status and the present of S. mutans (P=0.028).

Table (4.5): Distribution of study population according to the marital status

Positive or negative for the presence of S. mutans Marital Patients status Chi- P Value Single Married Widowed Divorced Square +ve(n=95) 10 82 2 1 % (24.4%) (33.1%) (25.0%) (33.4%) -ve(n=205) 31 166 6 2 % (75.6%) (66.9%) (75.0%) (66.6%) Total(n=300) 41(100%) 248(100%) 8(100%) 3(100%) 13.153 0.028 Control Single Married Widowed Divorced Total(n=300) 100 179 15 6 (100%) (33.0%) (60.0%) (5.0%) (2.0%)

4.6 Distribution of study population according to the phases of education

Table (4.6) shows the presence of S. mutans according to the phases of education. Thirty three (35.1%) of primary education phase patients had S.

mutans in their dental caries and saliva, while 61(64.9%) patients of this group were negative for S. mutans on saliva and dental caries.

Thirty six patients (31.0%) of secondary education phase had S. mutans in their dental caries and saliva, while 80 (69.0%) of secondary education phase patients were negative for S. mutans on saliva and dental caries.

Twenty six patients (28.9%) of higher education phase had S. mutans in their dental caries and saliva, while 64(71.1%) of higher education phase patients were negative for S. mutans on saliva and dental caries.

Table (4.6) shows that there were statistically significance between the education level and the present of S. mutans (P=0.001).

Table (4.6): Distribution of study population according to the phases of education

Positive or negative for the presence of S. mutans Education Patients Chi-Square P Value Primary Secondary Higher +ve(n=95) 33 36 26 % (35.1%) (31.0%) (28.9%) -ve(n=205) 61 80 64 % (64.9%) (69.0%) (71.1%) Total(n=300) 94(100%) 116(100%) 90(100%) 14.910 0.001 Control Primary Secondary Higher Total(n=300) 43 77 180 (100%) (14.3%) (25.7%) (60.0%)

4.7 Distribution of study population according to the accommodation

Table (4.7) shows the presence of S. mutans according to the accommodation. Forty nine (30.6%) of patients living in urban had S. mutans in their dental caries and saliva, while 111(69.4%) of patients living in urban were negative for S. mutans on saliva and dental caries.

Forty six (32.9%) of patients living in rural had S. mutans in their dental caries and saliva, while 94 (67.1%) of patients living in rural were negative for S. mutans on saliva and dental caries.

Table (4.7) shows that there were statistically not significance between the accommodation and the present of S. mutans (P=0.071).

Table (4.7): Distribution of study population according to the accommodation

Positive or negative for the presence of S. mutans Accommodation Patients Chi-Square P Value Urban Rural +ve(n=95) 49 46 % (30.6%) (32.9%) -ve(n=205) 111 94 % (69.4%) (67.1%) Total(n=300) 160(100%) 140(100%) 0.450 0.071 Control Urban Rural Total(n=300) 163 137 (100%) (54.3%) (45.7%)

4.8 Distribution of study population according to the smoking status

Table (4.8) shows the presence of S. mutans according to the smoking status.

Thirty five (29.1%) of smokers patients had S. mutans in their dental caries and saliva, while 85 (70.9%) of smokers participants living in urban were negative for S. mutans on saliva and dental caries.

Sixty (33.4%) of non-smokers patients had S. mutans in their dental caries and saliva, while 120(66.6%) of non-smokers patients were negative for S. mutans on saliva and dental caries.

Table (4.8) shows that there were statistically not significance between the smoking status and the present of S. mutans (P=0.089).

Table (4.8): Distribution of study population according to the smoking status

Positive or negative for the presence of S. mutans Smoking Patients Chi-Square P Value status Smoker Non smoker +ve(n=95) 35 60 % (29.1%) (33.4%) -ve(n=205) 85 120 % (70.9%) (66.6%) Total(n=300) 120(100%) 180(100%) 0.071 0.089 Control Smoker Non smoker Total(n=300) 106 194 (100%) (35.3%) (64.7%)

4.9 Distribution of study population according to the number of lost teeth

Table (4.9) shows the presence of S. mutans according to the number of lost teeth. Two (8.7%) of patients non-lost teeth had S. mutans in their dental caries and saliva, while 21(91.3%) of patients non-lost teeth were negative for S. mutans on saliva and dental caries.

Thirty nine (33.0%) of patients lose one tooth had S. mutans in their dental caries and saliva, while 79(67.0%) of patients lose one tooth were negative for S. mutans on saliva and dental caries.

Thirty (31.0%) of patients lost two teeth had S. mutans in their dental caries and saliva, while 67(69.0%) of patients lost two teeth were negative for S. mutans on saliva and dental caries.

Twenty four (38.7%) of patients lost more than two teeth had S. mutans in their dental caries and saliva, while 38(61.3%) of patients lost more than two teeth were negative for S. mutans on saliva and dental caries.

Table (4.9) shows that there were statistically significance between the number of lost teeth and the present of S. mutans (P=0.001).

Table (4.9): Distribution of study population according to the number of lost teeth

Positive or negative for the presence of S. mutans No. of lost Patients teeth Non One Two More Chi- P Value than two Square +ve(n=95) 2 39 30 24 % (8.7%) (33.0%) (31.0%) (38.7%) -ve(n=205) 21 79 67 38 % (91.3%) (67.0%) (69.0%) (61.3%) Total(n=300) 23(100%) 118(100%) 97(100%) 62(100%) 80.500 0.001 Control Non One Two More than two Total(n=300) 216 69 14 1 (100%) (72.0%) (23.0%) (4.7%) (0.3%)

4.10 Distribution of study population according to the types of toothpaste

Table (4.10) shows the presence of S. mutans according to the types of toothpaste used. Four (26.7%) of patients used synsodyne had S. mutans in their dental caries and saliva, while 11(73.3%) of patients used synsodyne were negative for S. mutans on saliva and dental caries.

Seven (31.8%) of patients used Colgate had S. mutans in their dental caries and saliva, while 15(68.2%) of patients used Colgate were negative for S. mutans on saliva and dental caries.

Six (19.3%) of patients used Paradontax had S. mutans in their dental caries and saliva, while 25(80.7%) of patients used Colgate were negative for S. mutans on saliva and dental caries.

Twenty five (28.7%) of patients used signal had S. mutans in their dental caries and saliva, while 62(71.3%) of patients used signal were negative for S. mutans on saliva and dental caries.

Five (50.0%) of patients used B.white had S. mutans in their dental caries and saliva, while 5(50.0%) of patients used B.white were negative for S. mutans on saliva and dental caries.

Forty eight (35.5%) of patients were not using toothpaste had S. mutans in their dental caries and saliva, while 87(64.5%) of patients were not using toothpaste were negative for S. mutans on saliva and dental caries.

Table (4.10) shows that there were statistically significance between the types of toothpaste used and the present of S. mutans (P=0.001).

Table (4.10): Distribution of study population according to the types of toothpaste

Positive or negative for the presence of S. mutans

Type of toothpaste Patients

Sensodyne Colgate Paradontax Signal B Non Chi- P Value White Square

+ve(n=95) 4 7 6 25 5 48

% (26.7%) (31.8%) (19.3%) (28.7%) (50%) (35.5%)

-ve(n=205) 11 15 25 62 5 87

% (73.3%) (68.2%) (80.7%) (71.3%) (50%) (64.5%)

Total 15 22 31 87 10 135

(n=300) (100%) (100%) (100%) (100%) (100%) (100%) 49.373 0.001

Control

Sensodyne Colgate Paradontax Signal B Non White

83 101 40 46 21 9 Total(n=300)

(100%) (27.7%) (33.7%) (13.3%) (15.3%) (7.0%) (3.0%)

4.11 Distribution of study population according to the number of times using tooth brushing

Table (4.11) shows the presence of S. mutans according to the number of times using tooth brushing. Forty eight (35.5%) of patients were not brushing teeth had S. mutans in their dental caries and saliva, while 87 (64.5%) of patients were not brushing teeth were negative for S. mutans on saliva and dental caries.

Twenty two (28.6%) of patients were brushing teeth once daily had S. mutans in their dental caries and saliva, while 55 (71.4%) of patients were brushing teeth once daily were negative for S. mutans on saliva and dental caries.

Twenty one (29.2%) of patients were brushing teeth twice daily had S. mutans in their dental caries and saliva, while 51 (70.8%) of patients were brushing teeth a twice daily were negative for S. mutans on saliva and dental caries.

Three (60.0%) patients were brushing teeth three times a day had S. mutans in their dental caries and saliva, while 2 (40%) of patients were brushing teeth three times a day were negative for S. mutans on saliva and dental caries.One (9.1%) patient was brushing teeth more than three times a day had S. mutans in their dental caries and saliva, while 10 (90.9%) of patients were brushing teeth more than three times a day were negative for S. mutans on saliva and dental caries.

Table (4.11) shows that there were statistically significance between the types of toothpaste used and the present of S. mutans (P=0.001).

Table (4.11): Distribution of study population according to the number of times using tooth brushing

No of times Positive or negative for the presence of S. mutans using tooth Patients brushing Non Once Twice Three More Chi- P Value daily daily daily times than 3 Square times +ve(n=95) 48 22 21 3 1 % (35.5%) (28.6%) (29.2%) (60.0%) (9.1%) -ve(n=205) 87 55 51 2 10 % (64.5%) (71.4%) (70.8%) (40.0%) (90.9%) Total 135 77 72 5 11 (n=300) (100%) (100%) (100%) (100%) (100%) Control 42.397 0.001 Non Once Twice Three More daily daily times than 3 times Total(n=300) 9 88 120 82 1 (100%) (3.0%) (29.4%) (40.0%) (27.3%) (0.3%)

4.12 Distribution of study population according to number of visits to the dental clinic yearly

Table (4.12) shows the presence of S. mutans according to the number of visits to the dental clinic yearly. Ten (52.6%) of patients were not visit the dental clinic had S. mutans in their dental caries and saliva, while 9 (47.4%) of patients were not visit the dental clinic were negative for S. mutans on saliva and dental caries.

Eighteen (25.7%) of patients were visit the dental clinic once yearly had S. mutans in their dental caries and saliva, while 52 (74.3%) of patients were visit the dental clinic once yearly were negative for S. mutans on saliva and dental caries.

Thirty (23.6%) of participants were visit the dental clinic twice yearly had S. mutans in their dental caries and saliva, while 97 (76.4%) of patients were visit

the dental clinic twice yearly were negative for S. mutans on saliva and dental caries.

Thirty seven (44.0%) of patients were visit the dental clinic more than two times yearly had S. mutans in their dental caries and saliva, while 47 (56.0%) of patients were visit the dental clinic more than two times yearly were negative for S. mutans on saliva and dental caries.

Table (4.12) shows that there were statistically significance between the times of visited the dental clinic and the present of S. mutans (P=0.001).

Table (4.12): Distribution of study population according to the times do you visit the dental clinic yearly

No of visits Positive or negative for the presence of S. mutans to dental Patients clinics Non One time Two times More Chi- P Value yearly than two Square times +ve(n=95) 10 18 30 37 % (52.6%) (25.7%) (23.6%) (44.0%) -ve(n=205) 9 52 97 47 % (47.4%) (74.3%) (76.4%) (56.0%) Total 19 70 127 84 (n=300) (100%) (100%) (100%) (100%) Control 61.730 0.001 Non One time Two times More than two times Total(n=300) 131 125 41 3 (100%) (43.6%) (41.7%) (13.7%) (1.0%)

4.13 Distribution of study population according to the brushing teeth before going to bed

Table (4.13) shows the presence of S. mutans according to the brushing teeth before going to bed. Twenty four (24.2%) of patients were brushing teeth before going to bed had S. mutans in their dental caries and saliva, while 75 (75.8%) of

patients were brushing teeth before going to bed were negative for S. mutans on saliva and dental caries.

Seventy one (35.3%) of patients were not brushing teeth before going to bed had S. mutans in their dental caries and saliva, while 130 (64.7%) of patients were not brushing teeth before going to bed were negative for S. mutans on saliva and dental caries.

Table (4.13) shows that there were statistically significance between the brushing teeth before going to bed and the present of S. mutans (P=0.001).

Table (4.13): Distribution of study population according to the brushing teeth before going to bed

Positive or negative for the Brushing teeth presence of S. mutans before sleeping Patients Chi-Square P Value Yes No +ve(n=95) 24 71 % (24.2%) (35.3%) -ve(n=205) 75 130 % (75.8%) (64.7%) Total 99 201 (n=300) (100%) (100%) Control 75.429 0.001 Yes No Total(n=300) 290 10 (100%) (96.7%) (3.3%)

4.14 Distribution of study population according to the types of mouthwash used

S. mutans according to the types of mouth wash using. All participants were not using mouth wash had S. mutans in their dental caries and saliva, while all participants were using mouth wash were negative for S. mutans on saliva and dental caries.

4.15 Culturing and Enumeration of S. mutans in 1ml of saliva in adult participants

A. Growth on Brain heart infusion agar The aim of culturing saliva sample from 10 random selected participants having dental caries on brain heart infusion agar was to enumerate fastidious and nonfastidious microorganisms, such as streptococci, pneumococci and meningococci. The total bacterial count was 3.5x106 CFUml-1 saliva (Table 4.14)

Table (4.14): Enumeration of bacteria in 1ml of saliva on brain heart infusion agar

mesaeurment 1st 2nd average 1 4.4x106 2.9x106 3.7x106 2 4.0x106 2.7x106 3.4x106 3 3.0x106 1.9x106 2.5x106 4 4.4x106 2.9x106 3.7x106 5 4.6x106 2.9x106 3.8x106 6 5.2x106 3.0x106 4.1x106 7 5.0x106 2.7x106 3.9x106 8 5.1x106 2.8x106 4.0x106 9 3.1x106 3.2x106 3.2x106 10 4.0x106 2.2x106 3.1x106 CFU/ml 3.5x106

B. Growth on Mitis Salivarius agar

According to colony morphology on mitis salivarius agar (Fig 3.1) we distinguish between different species of streptococcus. Our target species (S. mutans) was enumerated and compared with other close related species (Table 4.15) .

Table (4.15): Enumeration of S. mutans and related species in 1ml of saliva in patients

S. mitis S. salivarius S. mutans Measurement 1st 2nd average 1st 2nd average 1st 2nd average 1 2.7x105. 4.0x105 3.4x105 1.2x105 2.5x105 1.9 x105 4.7x105 1.0x106 7.4 x105 2 3.0x105 5.1x105 4.1x105 1.3x105 2.2x105 1.8 x105 4.3x105 1.2x106 8.2 x105 3 2.1x105 3.3x105 2.7x105 9.0x105 1.6x105 1.3 x105 3.1x105 8.2x105 5.7 x105 4 3.2x105 3.0x105 3.1x105 3.0x105 1.7x105 2.4 x105 4.8x105 4.0x105 4.4 x105 5 3.0x105 3.3x105 3.4x105 1.2x105 2.0x105 1.6 x105 4.5x105 3.8x105 4.2 x105 6 3.2x105 3.8x105 3.8x105 1.6x105 2.2x105 1.9 x105 4.7x105 3.9x105 4.3 x105 7 4.2x105 5.1x105 5.1x105 1.2x105 1.8x105 1.5 x105 4.4x105 1.0x105 2.7 x105 8 3.5x105 3.4x105 3.4x105 1.8x105 2.2x105 2.0 x105 4.8x105 1.0x105 2.9 x105 9 4.4x105 3.7x105 3.7x105 1.1x105 1.8x105 1.5 x105 3.2x105 1.5x105 2.4 x105 10 3.0x105 3.5x105 3.9x105 1.3x105 2.5x105 1.9 x105 3.5x105 6.6x105 5.1 x105 CFU/ml 3.6x105 1.8x105 4.7x105

S. salivarius 17.8%

S.mutans 46.5%

S. mitis 35.7%

Figure (4.1): Percentages of S. mutans , S. mitis and S. salivarius in 1ml of saliva taken from adult patients.

4.16 Results of biochemical tests for suspected S. mutans isolates

Biochemical results confirmed that the suspected S. mutans colonies isolated from mitis salivarius agar were gram-positive streptococci; positive for Voges proskaur test, and able to ferment sugars (Mannitol, Sucrose and Sorbitol). The colonies showed adhesive growth on the walls of the test tubes when ferment sucrose. They showed also alpha (a) hemolysis on the blood agar plates (Table 4.16)

Table(4.16): Results of biochemical tests for suspected S. mutans isolates

Biochemical tests Reaction of suspected S. mutans isolates Gram Stain Gram Positive, Streptococci Voges proskaur Test +ve Fermentation of Sucrose +ve Fermentation of Mannitol +ve Fermentation of Sorbitol +ve Hemolysis on blood agar Partial hemolysis (alpha hemolysis) Esculin hydrolysis +ve Catalase test -ve

4.17 Antimicrobial susceptibility

Table (4.17) shows the antimicrobial susceptibility for the most common used antibiotics against S. mutans. Vancomycin, doxycyclin, tetracycline, ciprofloxacin and amoxyclav were the most effective antibiotic respectively.

Table( 4.17): Antibiotics susceptibility for S. mutans

Antibiotics Sensitive (S) Intermediate (I) Resistant (R) No. % No. case % No. case % case Ampicillin (AMP) 1 1.1 6 6.3 88 92.6 Amoxicillin (AM) 7 7.4 0 0 88 92.6 Amoxyclav (AMC) 72 75.8 0 0 23 24.2 Ciprofloxacin (CIP) 77 81.1 2 2.1 16 16.8

Clindamycin (CD) 58 61.1 3 3.2 34 35.8 S. S. mutans

Doxycyclin (DO) 80 84.2 2 2.1 13 13.7 Erythromycin (E) 19 20.0 1 1.1 75 78.9 Oxacillin (OX) 3 3.2 0 0 92 96.8 Tetracycline (TE) 79 83.2 3 3.2 13 13.7 Vancomycin (VA) 95 100 0 0 0 0

100 90 80 70 60 50 S 40 I

30 R Percentage

20 10 0 AMP AMX AMC CIP DO E OX TE VA DA Antibiotics

Figure (4.2): Antibiotics susceptibility for S. mutans

Figure (4.3) and Figure (4.4) showed the inhibition zone caused by various antibiotics.

Figure(4.3): A photograph showing disk diffusion for ampicillin, tetracycline, ciprofloxacin, clindamycin and amoxicillin against S. mutans

Figure(4.4): A photograph showing disk diffusion for vancomycin, erythromycin, amoxyclav, oxacillin and doxycyclin against S. mutans

4.18 Antimicrobial activity of essential oils against three isolates of S. mutans

Nine essential oils were evaluated for their antimicrobial activity against 3 selected isolates of S .mutans (X, Y and Z). Table (4.18) shows the different types of oils and their measured inhibitory effect (in mm) on S. mutans.

Figure (4.5) illustrate an example of the inhibitory effect of clove bud oil against S. mutans.

Control CLOVE BUD

Figure (4.5): Clove bud against S. mutans

Table (4.18): Antimicrobial effect of different plant oils on S. mutans.

Name of plant oil S. mutans isolates Diameter of growth of inhibition zones ( mm ) Clove bud x 29 y 30 z 32

x 25 Tea Tree y 22 z 21 x 18 Sage y 22 z 19 x 30 Cinnamon Citratus y 28 z 27 x 16 Peppermint y 15 z 19 x 16 Ginger y 12 z R x 18 Cinnamon leaf y 20 z 19 x 18 Thyme y 21 z 16 x 18 Rosemary y 22 z R

15 X Y 10

Zone Zone of ihibition(mm) Z 5

Figure (4.6): Antimicrobial activity of essential oils against S. mutans

4.19 Efficacy of toothpaste against three isolates of S .mutans

The antimicrobial activity of different toothpaste against S. mutans was examined. Seen table (4.19) and figure (4.7).

Table (4.19): Antimicrobial activity of some toothpastes against S. mutans

Name of toothpaste S. mutans isolates Diameter of growth of inhibition zones ( mm ) x 4 B- White y 6 z R x 26 Colgate y 30 z 30 x 30 Signal y 20 z 20 x 28 Paradontax y 12 z 20 x 20 Synsodyne y 12 z 10

35 30

20 X 15 Y 10 Z 5 0

B. white Colgate Signal Paradontax Synsodyne Zone Zone of inhibition (mm) Tooth paste

Figure(4.7):Inhibitory effect of some tooth paste against S. mutans

4.20 Antimicrobial activity of mouth wash against S. mutans

This section deals with the results of inhibitory growth effect of some mouthwashes against S. mutans. Table (4.20) and figure (4.8) shows the inhibitory effect of mouth wash on S. mutans.

Table (4.20): Inhibitory effect of some mouth washes on the growth of 3 selected S. mutans isolates.

Diameter of growth of Name of mouth wash S. mutans isolates inhibition zones ( mm ) x 10 Garosept y 10 z 18 x 20 Gargarol y 30 z 30 x 20 Septoral y 10 z 30 x 10 Iodocare y 10 z 20

20 X 15 Y

10 Z Zone Zone of inhibition (mm) 5

0 Garosept Gargarol Septoral Iodocare Mouth washes

Figure (4.8): Mouthwashes and their effects on S. mutans

4.21 MIC and MBC determination

4.21.1 MIC and MBC of nine essential oils (μl/ml) Table 4.21.1and figure 4.9 shows the essential oils which exhibited antibacterial activity, were further determined for MIC and MBC values. The essential oils demonstrated MIC values ranging from 100 to 1500 μl/ml, and their MBC values was recorded less than 100 to 500 μl/ml. For Rosemary oil the result showed that MIC was 1500 μl/ml which mean MBC will be higher than1500 μl/ml so it was non effective for using as antibacterial agent.

Table (4.21): MIC of all the nine extracts against S. mutans

S. mutans Essential oils MIC MBC

Clove bud 100 200

Tea Tree 200 300

Sage 100 200

C.Citratus 500 500

Peppermint 500 500

Cinnamon leaf 100 100

Thyme 500 500

Rosemary 1500 -

Ginger 100 200

1600

1400 1200 1000 800 600 MIC 400 MBC Conc.of oils essential 200 0

Figure (4.9): MIC and MBC of essential oils against S. mutans

Chapter 5 Discussion

5.1 Clinical data and presence of S. mutans

Tooth decay (dental caries) is a significant health problem worldwide. It affects not only the vast majority of adults but also children (Marinho et al., 2013). Our study demonstrate the finding that S. mutans was more common in the age group 20-35 years (33.1%). A study conducted in Quebec Canada found that level of caries experience is very high in Quebec adults aged 35 to 44 (Brodeur et al., 2000). Another study performed by National Institutes of Health (NIH) on adults found that 92% of adults 20 to 64 years had dental caries in their permanent teeth (NIH, 2008). We found that the majority of positive cases for the presence of S. mutans in dental caries and saliva were detected in female patients. Such result was also achieved by Lukacs and largespada 2006 as well as by Pannu et al., 2013. According to the marital status, in this study we found that the divorced and married patients were positive for presence of S. mutans and dental carries more than other patients. A study was conducted in India by Jagadeesan et al., 2000 found that married female were positively correlated with caries (Jagadeesan et al., 2000). This result fit with a study conducted in Turkey by which the rate of positive significant caries index for single people was significantly lower than that of married people (Namal et al., 2008).

Distinctive but expected was the finding that the majority of positive cases for S. mutans were measured in non-smoker patients. From early reports in literature and in accordance with common belief, smoking was thought to actually help to reduce dental caries (Gibbs, 1952). Smoking increases thiocyanate level in saliva. Thiocyanate, a normal constituent of saliva, was found to have caries inhibiting effect (Reibel 2003; Johnson and Bain, 2000). Many studies confirm that brushing teeth twice a day or more would confer greater caries reductions than brushing once a day or less (Peros et al., 2012, Chesters1992; O’Mullane 1997; Chestnutt 1998; Ashley 1999). We have

achieved such results: Most negative cases for the presence of S. mutans were detected in patients who did use toothbrush and toothpaste one, twice and more than 3 times daily in order to clean their teeth. In this study, we found that the most positive cases for the presence of S. mutans in dental caries and saliva were detected in primary education phase patients. The effects of education level on the incidence of oral diseases have been assessed in many studies and such studies revealed different education level might contribute to a difference in knowledge, attitude and response of the patient to dental problems. One of the possible reasons that oral health status for educated people is better than others because these groups of people are more informed about their oral health needs and are also more likely to seek dental treatment. In addition, educated persons are expected to be able to afford dental service and should have better access to adequate dental care, and have better than average oral health habits (Sutcliffe 1996, Stahlnacke et al., 2003, Ekanayke and Perera, 2005).

The most positive cases for the presence of S. mutans in dental caries and saliva in our study were detected in patients who have loss of teeth, this mean that the S. mutans is the major cause of losing teeth among these patients. The persistent pH drop after exposure to fermentable dietary carbohydrates the metabolic activity of increased numbers of bacteria on the tooth surfaces or the increased presence of a particularly efficient carbohydrate fermenter such as S. mutans or S. sobrinus in the plaque. In either case, the tooth begins to lose some of its mineral content (Robyt and Martin 1983). The pH at which this demineralization begins is known as the critical pH and is near pH 5.0 to 5.5, which finally lead to teeth decaying and losing (Gibbons et al., 1986).

5.2 Enumeration of S. mutans and other oral streptococci Despite the conventional methods used in this work to enumerate total bacterial number and the count of S. mutans in saliva but the archived results fit in acceptable manner with the counts accomplished in other studies; The total bacterial count in saliva ranged between 106 CFU/ml (Gallez et al., 2000 and Ogawa et al., 2012) and 109 CFU/ ml (Amoroso et al., 2015). We demonstrate in this work a total bacterial count of 3.5x106 CFU/ml of saliva. We detected 95 patients from 300, having S. mutans in their saliva (32%). The average number of S. mutans was 4.7x105 CFU/ml. This count was around the count achieved in previously studies which range between 1x104 to 2x106 (Gu et al., 2002) or 1x105 to 1x106 (Pannu et al., 2013). In other previous studies, S. mitis and S. salivarius were commonly detected in saliva (Amoroso et al., 2015, Ogawa et al., 2012 and Kang et al., 2006); we detected the presence S. mitis and S. salivarius and have found that S. mutans was more prevalent in our target patients than S. mitis and S. salivarius. Our finding differ from those accomplished by Amoroso et al., 2015 that demonstrated a more prevalence of S. salivarius toward S. mutans.

5.3 Antibacterial agents and S. mutans Our results indicate that S. mutans have been found to be most susceptible against vancomycin (100%), tetracycline (83.2%), doxycycline (84.2%), ciprofloxacin (81.1%) and amoxyclav (75.8%). Dentists commonly prescribe most of the antibiotics employed in this study. A study conducted by Jubair 2015 indicated that most S. mutans isolates were resistance to amoxicillin and erythromycin with 90% and 78% from all isolates (Jubair 2015). In addition, this result was agreed with (Muna, 2011) in which found rate resistance for each amoxicillin and erythromycin was 87.5%. (El Sherbiny 2014) found that rate resistance to tetracycline 35% from all S. mutans isolates. This difference may be due to the influence of many factors like the age of the patient, the

season of samples collection and the frequently and uncontrolled use of antibiotics in Gaza strip.

5.4 Essential oils and its effect on S. mutans Agar diffusion tests are often used as qualitative methods to determine whether a bacterium is resistant, intermediately resistant or susceptible. However, the agar diffusion method can be used for determination of MIC values provided the necessary reference curves for conversion of inhibition zones into MIC values are available. After an agar plate is inoculated with the bacteria, a tablet, disk or paper strip with the antimicrobial agent is placed on the surface. During incubation the antimicrobial agent diffuses into the agar and inhibits growth of the bacteria if susceptible. Diffusion tests are cheap compared to most MIC- determination methods, but has been developed to give an approximate MIC- value.

Well standardized methods are essential for all kinds of susceptibility testing, since the methods are highly sensitive to variations in several factors, such as size of inoculum, contents and acidity of the growth medium, time and temperature of incubation. The agar diffusion methods are also strongly influenced by factors, such as agar depth, diffusion rate of the antimicrobial agent and growth rate of the specific bacteria.

The MIC-determination and disk diffusion methods described in this protocol are in accordance with the international recommendations given by the Clinical and Laboratory standard Institute (CLSI). The CLSI describes how to perform the tests and sets international guidelines for interpretation of the results. It should be noted that the WHO does not prescribe any specific method for performance and interpretation of susceptibility tests.

Internal quality control should be regularly performed as recommended by CLSI (CLSI 2011). 63

In our study 9 essential oils have been tested for in-vitro antimicrobial activity and some have demonstrated to be possessing potential antimicrobial activity. The effect of essential oils against S. mutans in our study shows that Clove bud, lemon grass and Tea tree were the most effective oils against S. mutans respectively. A study was conducted by Chaudhari et al., showed that Cinnamon oil was the most effective against S. mutans followed by lemongrass oil, cedar wood oil and clove bud oil (Chaudhari et al., 2012).

5.5 Tooth paste and mouth rinses and their effects on S. mutans The results of our study showed that different toothpaste brands exhibited wide range of inhibitory activity against the 3 tested isolates of S. mutans. Among all the investigated toothpastes, Colgate toothpaste has emerged as the most effective formulation compared to all other toothpaste formulations (with inhibition zones ranged from 25 to 30 mm) followed by Signal, Paradontax and Sensodyne. The lowest inhibitory effect were expressed by B white toothpaste with inhibition zones ranging from 0 to 5 mm. The mean inhibition zone diameters of the five toothpaste brands at full strength ranged between 5 and 30 mm. Colgate toothpaste emerged as the most effective against all the three isolates tested, this may be due to presence of sodium mono fluorophosphate and sodium fluoride. Signal toothpaste has significant antimicrobial activity against the tested organisms. It contains sodium mono fluorophosphate and calcium glycerophosphate as active ingredients. Prasanth 2011, demonstrate the fact that toothpastes containing mono fluorophosphates have inhibitory effect not only on S. mutans but also on oral pathogens like E. coli and C. albicans. Fluorides are popularly used in many oral health products as are reported to help in caries prevention (Marinho 2009). Sensodyne toothpaste had low inhibitory effect on tested pathogens that may be due to the presence of a single ingredient in its formulation (strontium chloride hexahydrate).

In case of mouth rinses, Gargarol was found to be the most effective mouth rinse, which showed maximum antimicrobial efficacy against the tested pathogens. This may be due to the presence of Chlorhexidine gluconate as major ingredient in its formulation; this observation fit with the results achieved by Prasanth 2011, which demonstrate that mouth rinse containing Chlorhexidine formulation has significant antimicrobial activity compared to those do not have this formulation.

Chapter 6 Summary, Conclusion and Recommendation

6.1 Summary 1. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in the age group 20-35 year.

2. the most positive cases for the presence of S. mutans in dental caries and saliva were detected in female patients.

3. Among the 95 positive cases, the presence of S. mutans in dental caries and saliva was detected in higher percentages in divorced and married patients.

4. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in primary education phase patients.

5. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in patients living in rural areas.

6. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in nonsmokers patients.

7. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in patients who have loss of teeth .

8. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in patients were not using any toothpaste or have used B white toothpaste.

9. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in the patients who were not visitors of the dental clinics .

6.2 Conclusion

1. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in female, divorced and married patients. 2. The most positive cases for the presence of S. mutans in dental caries and saliva were detected in patients who have loss of teeth. 3. The presence of S. mutans in dental caries and saliva were detected in patients who did not brush their teeth or using any toothpaste (with exception of B white). 4. In present study, we observed that the S. mutans were moderately resistant to antibiotics. 5. S. mutans isolated from patient from Gaza strip were resistant to significant range of antibiotics. The regular use of toothpaste is better for prevention rather than use of antibiotic reported in present study.

6.3 Recommendation

1. Brushing teeth at least twice a day with a fluoride-containing toothpaste. Preferably, brush after each meal and especially before going to bed.

1. Rinse daily with a fluoride or Chlorhexidine gluconate containing mouthwash. 2. Essential oils like Lemon grass, Clove bud and Tea tree can be used as antibacterial supplement in our region. 3. Use of plant extracts (essential oils) may be recommended as an supportive or alternative option to conventional formulations (toothpaste or mouth rinse) as these plant extracts may provide inexpensive, safe, effective and readily available alternative in maintaining oral hygiene. Lemon grass, Clove bud and Tea tree possess the best antibacterial activity.

Chapter 7 References

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Annexes

Annex (1)

Table (1)

اخي الكزين/ اختي الكزيوت...

ارجىا هٌكن الوسبعذة في تعبئت هذا االستبيبى والذي يهذف الً جوع بيبًبث هي الوزضً لهذف بحثً حىل هىضىع دراست عشل البكتيزيب الوسببت لتسىص االسٌبى وهذي استجببتهب للوضبداث الحيىيت ,شبكزيي لكن حسي تعبوًك.

Sociodemographic data

...... Name

...... Age

Male Female Gender

Single Married Widowed Divorced Marital status

Primary Secondary Higher education

Education

Urban Rural

Accommodation

Smoker Non smoker Smoking

اًب الوىقع/ة ادًبٍ اوافق علً اعطبء البيبًبث السببقت الخبصت بي الالسهت إلتوبم الذراست.

التىقيع------82

Table ( 1 )

Clinical Data

Male Female Gender

Pregnant Not pregnant If female

No. of lost teeth ......

No. of times of using ...... tooth brushing

Type of tooth paste ...... used

How many times do you visit the dental ...... clinic yearly

Yes No Brushing teeth before going to bed

اًب الوىقع/ة ادًبٍ اوافق علً اعطبء البيبًبث السببقت الخبصت بي الالسهت إلتوبم الذراست.

التىقيع------

Annex ( 2 )

Table ( 2 ) For doctor :

What type of mouth wash do you take ......

Amoxicillin Doxycycline

Amoxicillin Clave Erythromycin

Ampicillin Gentamycin

Azithromycin Levofloxacin

Ceftriaxone Methicillin

Cefuroxime Nalidixic Acid

Cephalexin Norofloxacine

Cefotaxime Ofloxacin

Antibiotic treatment Cefadroxil Oxacillin

Cephalotin Penicillin

Cefaclor Piperacillin

Ceftazidime Rifampicin

Cefixime Tetracycline

Cefazolin Vancomycin

Chloramphenicol

Ciprofloxacin

Cloxacillin

Co-trimoxazole